The antioxidant effect of tannic acid on the in vitro copper-mediated formation of free radicals
Section snippets
Reagents
Ascorbic acid, catalase, deferoxamine mesylate (DFO), 2-deoxyribose, EDTA, ellagic acid, gallic acid, rutin, tannic acid (TA), and thiobarbituric acid (TBA) were purchased from Sigma (St. Louis, MO). Other reagents were of analytical grade. Stock solutions of gallic acid, rutin, TA (1 mM), and ellagic acid (see Table 1) were freshly prepared in 20 mM phosphate buffer (pH 7.2). Pyridoxal isonicotinoyl hydrazone (PIH, donated by Dr. Prem Ponka, Montreal), copper sulfate, and TBA stock solutions
Copper-mediated 2-deoxyribose degradation
Preliminary studies determined the time course of oxidative damage to 5 mM 2-deoxyribose induced by 5 μM Cu(II) and 0.5 mM ascorbate. The addition of 10 μM TA at time zero of incubation, i.e., before the addition of ascorbate, caused a marked inhibition of 2-deoxyribose degradation. The rate of 2-deoxyribose degradation was linear for approximately 10 min in the absence or in the presence of TA (data not shown; n = 3). Therefore, we selected an 8 min incubation period for the following experiments.
A
Conclusions
We have demonstrated that TA inhibits OH formation and, thus, 2-deoxyribose degradation induced by the ascorbate/Cu(II)/O2 system. Since TA inhibits Cu(II)-mediated ascorbate oxidation, it is possible that the “pathway” of OH formation (Eqs. (1), (2), (3), (4), (5), (6)) is halted by TA in its first step (Eq. (1)). The complexation of Cu(II) by TA (see Fig. 9 and [45]; the complex may be converted to Cu(I)n–TA(oxidized)[22]) may inhibit the reaction of Cu(II) with ascorbate. Thus, we can
Acknowledgments
We thank Antonieta C.R. Alencastro (Universidade de Brasília (UnB)), Francisco Alves (UnB), Núbia Shelli (formerly at UnB), Alice A. Mota (formerly at UnB) and Beatrice Allain (São Carlos, Brazil) for their technical assistance, Dr. Fernando Pellegrini (Universidade Federal de Goiás) for the use of the EPR spectrometer, and Dr. Fernando Dutra (USP, Brazil) for relevant discussions and some of the O2 uptake determinations. We are also indebted to one of the anonymous referees for helping us gain
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- 1
Currently at Law School, Universidade de Brasília, Brazil.
- 2
Currently at EMPRAPA-Planaltina, Brasília, Brazil.